Radiolabeled GRPR-Antagonists for Diagnostic Imaging and Treatment of GRPR-Positive Cancer

ABSTRACT

The present invention relates to probes for use in the detection, imaging, diagnosis, targeting, treatment, etc. of cancers expressing the gastrin releasing peptide receptor (GRPR). For example, such probes may be molecules conjugated to detectable labels which are preferably moieties suitable for detection by gamma imaging and SPECT or by positron emission tomography (PET) or magnetic resonance imaging (MRI) or fluorescence spectroscopy or optical imaging methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/817,776 filed Nov. 20, 2017, which is a continuation of U.S. patentapplication Ser. No. 14/431,096 filed Sep. 25, 2013, now U.S. Pat. No.9,839,703, which is a U.S. National Phase Patent Application ofPCT/US2013/061712 filed Sep. 25, 2013 which claims priority of U.S.Provisional Patent Application No. 61/705,513 filed Sep. 25, 2012, whichare hereby incorporated herein by reference in their entireties.

INCORPORATION OF SEQUENCE LISTING

The content of the electronically submitted sequence listing in theASCII text file (Name: GRPR SubstituteSequenceListing_ST25.txt; Size:2439 bytes; and Date of Creation: Sep. 19, 2019) filed with theapplication is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Cancer cells have been shown to express a variety of specificbiomolecules such as peptide-receptors, which may serve as recognitionsites for a wide range of circulating vectors, as for examplepeptide-ligands. In case the expression of the target-receptor is higheron malignant cells than in surrounding healthy tissue, the opportunityarises to exploit the interaction between these two molecular entities.For diagnostic imaging or targeted therapy applications, a naturalpeptide-ligand could be modified to stably bind a diagnostic or atherapeutic radionuclide, e.g. a radiometal or a radiohalogen.

In many cases, a bifunctional chelator is covalently coupled via acarboxyl-functionality to the N-terminal amine of the peptide-ligand toform a peptide bond. In order to increase the biological stability,hydrophilicity, receptor binding affinity and/or internalizationefficacy, further modifications of native receptor ligands areattempted, such as strategic amino acid replacements in the peptidechain. Alternatively, introduction of suitable spacers between thechelator and the peptide receptor recognition site or hetero/homopeptide-multimerization may equally lead to advantageous improvements ofmany biological parameters eventually improving overall pharmacokineticsand target accumulation of the radioactive probe.

The resulting peptide-chelate conjugate after labeling with a diagnosticor a therapeutic radionuclide (radiopeptide) is administered to thepatient. The radiopeptide selectively accumulates on cancer-site(s)through specific interaction with the target-molecule, i.e. its cognatepeptide-receptor, highly expressed on the tumor. In case of a diagnosticradionuclide, the tumor and metastases are then localized by imaging thesite(s) where the radioactive decay occurs using an external imagingdevice. When the peptide-chelate conjugate is labeled with a therapeuticradionuclide, a radiotoxic load is delivered specifically to the primarytumor and its metastases. The therapeutic radionuclide will then decayon the cancer site(s), releasing corpuscular energy to kill or to reduce(the growth of) the lesions.

This strategy has been elegantly exploited in the area of somatostatinand its receptors. The latter are abundantly expressed in a variety ofhuman tumors, and especially in neuroendocrine tumors (NETs). The adventof OctreoScan® ([¹¹¹In-DTPA]octreotide) in clinical practice for thesuccessful diagnostic imaging of NETs was soon followed by many newimproved somatostatin analogs labeled with a wide range of medicallyrelevant radiometals useful not only for conventional imaging with agamma-camera, but also for PET and, most importantly, for radionuclidetherapy. Ongoing clinical trials have revealed the therapeutic efficacyof these new radiopeptides.

Peptide-receptors and their ligands have emerged as attractive moleculartools in cancer diagnosis and therapy. For example, high densityexpression of gastrin releasing peptide receptors (GRPRs) has beendocumented in several frequently occurring human tumors, such as inprostate cancer, mammary carcinoma and lung cancer. As a consequence,GRPRs have lately been gaining momentum as preferred molecular targetsfor radiolabeled bombesin-like peptides with the aim to upgrade thediagnostic and therapeutic arsenal of nuclear oncology.

Bombesin (BBN) is a tetradecapeptide initially isolated from the skin ofthe European frog Bombina bombina. Bombesin and its related peptidesaffect thermoregulation and food-intake after binding to specificreceptors in humans. These receptors comprise three subtypes in mammals,the neuromedin B receptor (NMBR or BB₁R) with a high affinity for NMB,the GRPR (or BB₂R) with a high affinity for GRP and the BB₃R, which isan orphan receptor with no-known ligand identified yet. Amphibian BBNbinds to NMBR and GRPR subtypes with a high affinity. NMB and GRP arethe mammalian counterparts of amphibian BBN and are all related instructure.

Most radiolabeled BBN-like peptides developed for molecular imaging andradionuclide therapy of human tumors have been based on native BBN, oron its C-terminal octapeptide fragment still able to bind the GRPR.These analogs modified as detailed above typically exhibit agonisticproperties and internalize in the intracellular region of malignantcells after binding to the GRPR. This property translates into a highaccumulation of the radiolabel in the GRPR⁺ lesions, thereby enhancingeither diagnostic sensitivity or therapeutic efficacy.

Unfortunately, BBN-like peptides are potent GRPR-agonists, elicitingadverse effects related to gastrointestinal motility andthermoregulation when intravenously (iv) administered in human even insmall amounts. In addition, BBN-like peptides are mitogenic. The aboveproperties have restrained the thorough clinical validation and/or theeventual commercial exploitation of a few promising agonist-basedradiolabeled bombesins. This is particularly relevant in the case oftargeted radionuclide therapy whereby higher peptide amounts need to beiv administered in patients.

Unlike radiolabeled BBN agonists, radiolabeled somatostatin-agonists,which internalize equally well into somatostatin receptor-expressingmalignant cells, do not elicit undesirable physiological effects afteriv injection in humans. This fact has fostered the extended andsystematic clinical validation of a few promising radiolabeledsomatostatins even in the domain of radionuclide tumor therapy.

The radiotracer ([^(99m)Tc]Demobesin 1,[^(99m)Tc—N₄′]DPhe-Gln-Trp-Ala-Val-Gly-His-Leu-NHEt) is known and usedin mice bearing human prostate cancer PC-3 xenografts, where[^(99m)Tc]Demobesin 1 showed exceptionally superior pharmacokineticproperties as opposed to similarly affine bombesin-based agonists, asfor example [^(99m)Tc]Demobesin 3-6. Besides its significantly highertumor accumulation, [^(99m)Tc]Demobesin 1 cleared very rapidly from thebody of mice and the pancreas, a strongly GRPR-positive organ.

Although first studies in a limited number of prostate cancer patientsverified the excellent tolerability of the radiotracer, they revealed asub-optimal pharmacokinetic profile in humans preventing a furtherexpanded clinical application as a diagnostic imaging tool. Morespecifically, [^(99m)Tc]Demobesin 1 despite its rapid body and pancreasclearance and its rather good in vivo stability, exhibited insufficientretention in malignant lesions in humans as compared to radiolabeledBBN-like agonists. Furthermore, [^(99m)Tc]Demobesin 1 was designed fordiagnostic imaging using conventional gamma camera or SPECT and isunsuitable for PET or radionuclide therapy applications. Althoughlabeling with the PET radionuclide ^(94m)Tc is feasible by means of theacyclic N₄-system, the medical use of this radionuclide is restrictedboth by sub-optimal nuclear characteristics and inconvenient production.On the other hand, therapeutic options are restricted to ^(186/188)Re,as the N₄-chelator cannot stably bind most of bi- and trivalentradiometals used in nuclear medicine.

It is therefore the object of the present invention to achieve highuptake and retention of a diagnostic and a therapeutic radiolabelselectively to GRPR⁺-cancer, both primary and metastatic.

SUMMARY OF THE INVENTION

The present invention relates to probes for use in the detection,imaging, diagnosis, targeting, treatment, etc. of cancers expressing thegastrin releasing peptide receptor (GRPR). Such probes may be moleculesconjugated to detectable labels which are preferably moieties suitablefor detection by gamma imaging and SPECT or by positron emissiontomography (PET) or magnetic resonance imaging (MRI) or fluorescencespectroscopy or optical imaging methods. Such probes may also bemolecules conjugated to anticancer drugs or to moieties containing atherapeutic radionuclide and are able to deliver a cytotoxic load suchas a cytotoxic drug or a therapeutic radionuclide at the site(s) ofdisease.

Certain embodiments of the invention are drawn to a GRPR-antagonist ofthe general formula:

MC—S—P

wherein:

at least one (radio)metal (M) and a chelator (C) which stably binds M;alternatively MC may represent a Tyr- or a prosthetic group carrying a(radio)halogen;

S is an optional spacer covalently linked between the N-terminal of Pand C and may be selected to provide a means for (radio)halogenation;P is a GRP receptor peptide antagonist of the general formula:

Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Z

Xaa₁ is not present or is selected from the group consisting of aminoacid residues Asn, Thr, Phe, 3-(2-thienyl)alanine (Thi),4-chlorophenylalanine (Cpa), α-naphthylalanine (α-Nal),β-naphthylalanine (β-Nal), 1,2,3,4-tetrahydronorharman-3-carboxylic acid(Tpi), Tyr, 3-iodo-tyrosine (o-I-Tyr), Trp, pentafluorophenylalanine(5-F-Phe) (all as L- or D-isomers);

Xaa₂ is Gln, Asn, His

Xaa₃ is Trp, 1,2,3,4-tetrahydronorharman-3-carboxylic acid (Tpi)

Xaa₄ is Ala, Ser, Val

Xaa₅ is Val, Ser, Thr

Xaa₆ is Gly, sarcosine (Sar), D-Ala, β-Ala

Xaa₇ is His, (3-methyl)histidine (3-Me)His

Z is selected from —NHOH, —NHNH₂, —NH-alkyl, —N(alkyl)₂, or —O-alkyl

or

wherein X is NH (amide) or 0 (ester) and R1 and R2 are the same ordifferent and selected from a proton, a (substituted)alkyl, a(substituted) alkyl ether, an aryl, an aryl ether or an alkyl-, halogen,hydroxyl or hydroxyalkyl substituted aromatic group.

In certain embodiments the GRPR-antagonist of the invention is asdescribed above and wherein Z is preferably selected from one of thefollowing formulae, wherein X is NH or O:

Further, in certain embodiments, the GRPR-antagonist is as describedabove and R1 is the same as R2.

In certain of any of the embodiments described above, the invention isdrawn to wherein P is selected from the group consisting of:

(SEQ ID NO: 1) DPhe-Gln-Trp-Ala-Val-Gly-His-NH-CH[CH₂-CH(CH₃)₂]₂;(SEQ ID NO: 2) DPhe-Gln-Trp-Ala-Val-Gly-His-O-CH[CH₂-CH(CH₃)₂]₂;(SEQ ID NO: 3) DPhe-Gln-Trp-Ala-Val-Gly-His-NH-CH(CH₂-CH₂-CH₂- CH₃)₂;(SEQ ID NO: 4) DTyr-Gln-Trp-Ala-Val-Gly-His-NH-CH[CH₂-CH(CH₃)₂]₂;

In certain of any of the embodiments described above, the invention isdrawn to wherein the radionuclide metal M or radiohalogen is suitablefor diagnostic or therapeutic use, in particular for imaging orradionuclide therapy and selected from the group consisting of ¹¹¹In,^(133m)In, ^(99m)Tc, ^(94m)Tc, ⁶⁷Ga, ⁶⁶Ga, ⁶⁸Ga, ⁵²Fe, ¹⁶⁹Er, ⁷²As,⁹⁷Ru, ²⁰³Pb, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ¹⁸⁶Re, ¹⁸⁸Re, ⁸⁶Y, ⁹⁰Y, ⁵¹Cr, ^(52m)Mn,¹⁵⁷Gd, ¹⁷⁷Lu, ¹⁶¹Tb, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁰⁵Rh, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁵³Sm, ¹⁴⁹Pm,¹⁵¹Pm, ¹⁷²Tm, ¹²¹Sn, ^(117m)Sn, ²¹³Bi, ¹⁴²Pr, ¹⁴³Pr, ¹⁹⁸Au, ¹⁹⁹Au,halogens: ¹²³I, ¹²⁴I, ¹²⁵I, ¹⁸F, a.o.

In certain of any of the embodiments described above, the invention isdrawn to wherein the metal chelator C is a metal chelator for di- andtrivalent metals.

In certain of any of the embodiments described above, the invention isdrawn to wherein the metal chelator for di- and trivalent metals is aDTPA-, NOTA-, DOTA-, or TETA-based chelator or a mono- or bifunctionalderivative thereof.

In certain of any of the embodiments described above, the invention isdrawn to wherein the metal chelator C is selected from the groupconsisting of:

In certain of any of the embodiments described above, the invention isdrawn to wherein the metal chelator C is a metal chelator for technetiumor rhenium.

In certain of any of the embodiments described above, the invention isdrawn to wherein C is selected from acyclic tetraamine-, cyclam-, PnAO-,or tetradentate chelators containing P₂S₂—, N₂S₂— and N₃S-donor atomsets and mono- and bifunctional derivatives thereof, orHYNIC/co-ligand-based chelators, or bi- and tridentate chelators formingorganometallic complexes via the tricarbonyl technology.

In certain of any of the embodiments described above, the invention isdrawn to wherein C is selected from the group consisting of:

In certain of any of the embodiments described above, the invention isdrawn to wherein the spacer S is linked between P and C by covalentbonds and may be selected to provide a means for (radio)iodination.

In certain of any of the embodiments described above, the invention isdrawn to wherein S is selected from the group consisting of:

a) aryl containing residues of the formulae:

wherein PABA is p-aminobenzoic acid, PABZA is p-aminobenzylamine, PDA isphenylenediamine and PAMBZA is p-(aminomethyl)benzylamine;b) dicarboxylic acids, ω-aminocarboxylic acids, α,ω-diaminocarboxylicacids or diamines of the formulae:

wherein DIG is diglycolic acid and IDA is iminodiacetic acid;c) PEG spacers of various chain lengths, in particular PEG spacersselected from the formulae:

c) α- and β-amino acids, single or in homologous chains of various chainlengths or heterologous chains of various chain lengths, in particular:

GRP(1-18), GRP(14-18), GRP(13-18), BBN(1-5), or [Tyr⁴]BBN(1-5); or

d) combinations of a, b and c.

In certain of any of the embodiments described above, the invention isdrawn a GRPR-antagonist selected from the group consisting of compoundsof the formulae:

wherein MC and P are as defined in any one of the preceding.

In certain embodiments of the invention is drawn to a GRPR-antagonist asdescribed in any of the above embodiments for use as a medicament.

In certain embodiments of the invention is drawn to a GRPR-antagonist asdescribed in any of the above embodiments for use as diagnostic ortherapeutic agent for detecting, diagnosing or treating primary and/ormetastatic GRPR⁺ cancer.

In certain embodiments of the invention is drawn to a GRPR-antagonist asdescribed in any of the above embodiments, wherein the cancer isselected from prostate cancer, breast cancer, small cell lung cancer,colon carcinoma, gastrointestinal stromal tumors, gastrinoma, renal cellcarcinomas, gastroenteropancreatic neuroendocrine tumors, oesophagealsquamous cell tumors, neuroblastomas, head and neck squamous cellcarcinomas, as well as in ovarian, endometrial and pancreatic tumorsdisplaying neoplasia-related vasculature that is GRPR⁺.

In certain embodiments of the invention is drawn to a GRPR-antagonist asdescribed in any of the above embodiments, wherein the cancer is a humancancer.

Certain embodiments of the invention are drawn to a therapeuticcomposition, comprising a GRPR-antagonist as described in any of theembodiments above and a therapeutically acceptable excipient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A. Shows the biodistribution of [¹¹¹In]NeoBOMB-1(¹¹¹In-DOTA-(p-aminobenzylamine-diglycolicacid)-[DPhe⁶,His¹²-CO—NH—CH[(CH₂CH(CH₃)₂)₂,des-Leu¹³,des-Met⁴]BBN(6-14))in female SCID mice bearing PC-3 tumors (hGRPR+).

FIG. 1B. Shows a radiochromatogram of ex-vivo mouse blood 5 min afterinjection of [¹¹¹In]NeoBOMB-1.

FIG. 1C. Shows the biodistribution of [^(T77)Lu]NeoBOMB-1(¹⁷⁷Lu-DOTA-(p-aminobenzylamine-diglycolicacid)-[DPhe⁶,His¹²-CO—NH—CH[(CH₂CH(CH₃)₂]₂,des-Leu¹³,des-Met¹⁴]BBN(6-14)) in female SCID mice bearing PC-3 tumors(hGRPR+).

FIG. 1D. Shows a radiochromatogram of ex-vivo mouse blood 5 min afterinjection of [¹⁷⁷Lu]NeoBOMB-1.

FIG. 1E. Shows the biodistribution of [⁶⁷Ga]NeoBOMB-1(⁶⁷Ga-DOTA-(p-aminobenzylamine-diglycolicacid)-[DPhe⁶,His¹²-CO—NH—CH[(CH₂CH(CH₃)₂]₂, des-Leu¹³, des-Met⁴]BBN(6-14)) in female SCID mice bearing PC-3 tumors (hGRPR+).

FIG. 1F. Shows a radiochromatogram of ex-vivo mouse blood 5 min afterinjection of [⁶⁷Ga]NeoBOMB-1.

FIG. 2A. Shows the biodistribution of [^(99m)Tc]NeoBOMB-2(^(99m)Tc—N₄-(p-aminobenzylamine-diglycolicacid)-[DPhe⁶,His¹²-CO—NH—CH[(CH₂—CH(CH₃)₂]₂, des-Leu¹³, des-Met¹⁴]BBN(6-14)) in female SCID mice bearing PC-3 tumors (hGRPR+).

FIG. 2B. Shows a radiochromatogram of ex-vivo mouse blood 5 min afterinjection of [^(99m)Tc]NeoBOMB-2.

DETAILED DESCRIPTION

The research leading to the invention has unexpectedly revealed analternative route for effective in vivo targeting ofsomatostatin-positive tumors, namely the use of somatostatin receptorantagonists. Most surprisingly and against their inability tointernalize, such analogs have shown a much higher uptake and retentionin animal xenografts and a very rapid washout from background tissues.

A tentative explanation for the higher tumor uptake of somatostatinreceptor antagonists is their ability to bind to a significantly highernumber of the overall somatostatin receptor population available on thecell-membrane of cancer cells than their internalizing agonisticcounterparts.

According to the invention, GRPR-antagonists are chemically modified toaccommodate a diagnostic and/or therapeutic radionuclide that theystably bind. After administration in a human or an animal subject theyserve as a molecular vehicle to transfer a radiodiagnostic signal and/ora radiotoxic load on the primary GRPR⁺-tumor and its metastases.

More specifically, it was found according to the invention thatadministration of certain novel GRPR-antagonist-based radioligandsunexpectedly resulted in an unprecedentedly high and specific uptake anda remarkably prolonged retention of human GRPR⁺-xenografts in mice incontrast to [^(99m)Tc]Demobesin 1. Furthermore, these agents showedsignificantly higher metabolic stability after injection in mice,compared to [^(99m)Tc]Demobesin 1.

The GRPR-antagonists of the invention have important structuraldifferences in relation to the original [^(99m)Tc]Demobesin 1 motif.Firstly, their labeling with a wide range of bi- and trivalentradiometals, but also with ^(99m)Tc and ^(186/188)Re, is made possibleby coupling of suitable bifunctional chelators at their N-terminus inaddition to tetraamine-related frameworks. In this way, radiodiagnosticimaging is possible with SPECT and PET with gamma and positron-emitterswhile labeling with beta-, Auger and alpha emitters is feasible as well,opening the opportunity for therapeutic applications. Then, theirmetabolic stability and pharmacokinetic profile, especially in terms oftumor-retention has largely improved, as demonstrated by preclinicalbiodistribution results in female SCID mice bearing human PC-3xenografts presented at length.

More specifically, the structure of new analogs comprises the followingparts:

a) The chelator attached to the N-terminus—this can be either an acyclicor a cyclic tetraamine, HYNIC, N₃S-chelators and derivatives thereof,linear or cyclic polyamines and polyaminopolycarboxylates like DTPA,EDTA, DOTA, NOTA, NOTAGA, TETA and their derivatives, a.o. In addition,a suitable group, such a prosthetic group or a Tyr, for labeling withradiohalogens, can be introduced at this position;

b) The radionuclide—this may be i) a gamma emitter, such as ^(99m)Tc,¹¹¹In, ⁶⁷Ga, ¹³¹I, ¹²⁵I, a.o., suitable for imaging with a conventionalgamma-camera, a SPECT or an hybrid SPECT/CT or SPECT/MRI system; ii) apositron emitter, such as ⁶⁸Ga, ⁶⁶Ga, ⁶⁴Cu, ⁸⁶Y, ⁴⁴Sc, ¹²⁴I, ¹⁸F, a.o.,suitable for imaging with a PET or a hybrid PET/CT or PET/MRI system, oriii) a beta, Auger or alpha emitter, such as ¹⁸⁶Re, ¹⁸⁸Re, ⁹⁰Y, ¹⁷⁷Lu,¹¹¹In, ⁶⁷Cu, ²¹²Bi, ¹⁷⁵Yb, ⁴⁷Sc, ¹³¹I, ¹²⁵I, etc., suitable forradionuclide therapy;

c) The spacer between the chelator and the peptide motif, which may varyin length, type and lipophilicity and may include PEGx (x=0-20), naturaland unnatural amino acids, sugars, alkylamino residues or combinationsthereof;

d) The peptide chain, with strategic amino acid replacements undertakenwith D-amino acids, unnatural amino acids and other suitable residues.

e) The C-terminus, wherein the both Leu¹³ and Met¹⁴-NH₂ in the nativeBBN sequence have been omitted. Terminal His¹² is present as theamidated or ester form, whereby amides or esters may be represented byseveral mono- and di-alkylamides, aromatic amides or mixed alkyl-arylamides, or alkyl and/or aryl esters.

The invention thus relates to GRPR-antagonists of the general formula

MC—S—P

wherein:MC is a metal chelate, which comprises:

-   -   at least one (radio)metal (M) and a chelator (C) which stably        binds M; alternatively MC may represent a Tyr- or a prosthetic        group carrying a (radio)halogen.        S is an optional spacer covalently linked between the N-terminal        of P and C and may be selected to provide a means for        (radio)halogenation;        P is a GRP receptor peptide antagonist of the general formula:

Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Z

wherein:

Xaa₁ is not present or is selected from the group consisting of aminoacid residues Asn, Thr, Phe, 3-(2-thienyl)alanine (Thi),4-chlorophenylalanine (Cpa), α-naphthylalanine (α-Nal),β-naphthylalanine (β-Nal), 1,2,3,4-tetrahydronorharman-3-carboxylic acid(Tpi), Tyr, 3-iodo-tyrosine (o-I-Tyr), Trp, pentafluorophenylalanine(5-F-Phe) (all as L- or D-isomers);

Xaa₂ is Gln, Asn, His

Xaa₃ is Trp, 1,2,3,4-tetrahydronorharman-3-carboxylic acid (Tpi)

Xaa₄ is Ala, Ser, Val

Xaa₅ is Val, Ser, Thr

Xaa₆ is Gly, sarcosine (Sar), D-Ala, β-Ala

Xaa₇ is His, (3-methyl)histidine (3-Me)His

Z is selected from —NHOH, —NHNH₂, —NH-alkyl, —N(alkyl)₂, or —O-alkyl

or

wherein X is NH (amide) or 0 (ester) and R1 and R2 are the same ordifferent and selected from a proton, a (substituted)alkyl, a(substituted) alkyl ether, an aryl, an aryl ether or an alkyl-, halogen,hydroxyl or hydroxyalkyl substituted aromatic group.

Z is preferably selected from one of the following formulae, wherein Xis NH or O:

Preferably, R1 is the same as R2.

In the GRPR-antagonists of the invention P is preferably selected fromthe group consisting of:

(SEQ ID NO: 1) DPhe-Gln-Trp-Ala-Val-Gly-His-NH-CH[CH₂-CH(CH₃)₂]₂;(SEQ ID NO: 2) DPhe-Gln-Trp-Ala-Val-Gly-His-O-CH[CH₂-CH(CH₃)₂]₂;(SEQ ID NO: 3) DPhe-Gln-Trp-Ala-Val-Gly-His-NH-CH(CH₂-CH₂-CH₂- CH₃)₂;(SEQ ID NO: 4) DTyr-Gln-Trp-Ala-Val-Gly-His-NH-CH[CH₂-CH(CH₃)₂]₂;

The radionuclide, a metal M or a halogen, is suitable for diagnostic ortherapeutic use, in particular for imaging or radionuclide therapy andpreferably selected from the group consisting of ¹¹¹In, ^(133m)In,^(99m)Tc, ^(94m)Tc, ⁶⁷Ga, ⁶⁶Ga, ⁶⁸Ga, ⁵²Fe, ¹⁶⁹Er, ⁷²As, ⁹⁷Ru, ²⁰³Pb,⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ¹⁸⁶Re, ¹⁸⁸Re, ⁸⁶Y, ⁹⁰Y, ⁵¹Cr, ^(52m)Mn, ¹⁵⁷Gd, ¹⁷⁷Lu,¹⁶¹Tb, ¹⁶⁹Yb, ¹⁷⁵Yb, l⁰⁵Rh, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁵³Sm, ¹⁴⁹Pm, ¹⁵¹Pm, ¹⁷²Tm,¹²¹Sn, ^(117m)Sn, ²¹³Bi, ¹⁴²Pr, ¹⁴³Pr, ¹⁹⁸Au, ¹⁹⁹Au, ¹²³I, ¹²⁴I, ¹²⁵I,¹⁸F a.o.

The metal chelator C is preferably a metal chelator for di- andtrivalent metals, and is in particular a DTPA-, NOTA-, DOTA-, orTETA-based chelator or a mono- or bifunctional derivative thereof.

Preferably, the metal chelator C is selected from the group consistingof:

When the metal chelator C is a metal chelator for technetium or rhenium,it is preferably selected from acyclic tetraamine-, cyclam-, PnAO-, ortetradentate chelators containing P₂S₂—, N₂S₂— and N₃S-donor atom setsand mono- and bifunctional derivatives thereof, or HYNIC/co-ligand-basedchelators, or bi- and tridentate chelators forming organometalliccomplexes via the tricarbonyl technology.

Suitable examples of C are:

The spacer S is linked between P and C by covalent bonds and may beselected to provide a means for using a radiohalogen, such as(radio)iodination. The spacer is preferably selected from the groupconsisting of:

a) aryl containing residues of the formulae:

wherein PABA is p-aminobenzoic acid, PABZA is p-aminobenzylamine, PDA isphenylenediamine and PAMBZA is p-(aminomethyl)benzylamine;b) dicarboxylic acids, ω-aminocarboxylic acids, α,ω-diaminocarboxylicacids or diamines of the formulae:

wherein DIG is diglycolic acid and IDA is iminodiacetic acid;c) PEG spacers of various chain lengths, in particular PEG spacersselected from the formulae:

d) α- and β-amino acids, single or in homologous chains of various chainlengths or heterologous chains of various chain lengths, in particular:

GRP(1-18), GRP(14-18), GRP(13-18), BBN(1-5), or [Tyr⁴]BBN(1-5); or

e) combinations of a, b and c.

GRPR-antagonists of the invention are preferably selected from the groupconsisting of compounds of the formulae:

wherein MC and P are as defined above.

It is understood that specific chemical structures disclosed herein areillustrative examples of various embodiments of the invention and thatGRPR-antagonists of the general formula: MC—S—P are not limited to thestructures of examples provided.

The invention further relates to a therapeutic composition, comprising aGRPR-antagonist as claimed and a therapeutically acceptable excipient.

The invention also relates to the GRPR-antagonists as claimed for use asa medicament. The medicament is preferably a diagnostic or therapeuticagent for diagnosing or treating primary and/or metastatic GRPR⁺cancers, such as prostate cancer, breast cancer, small cell lung cancer,colon carcinoma, gastrointestinal stromal tumors, gastrinoma, renal cellcarcinomas, gastroenteropancreatic neuroendocrine tumors, oesophagealsquamous cell tumors, neuroblastomas, head and neck squamous cellcarcinomas, to name some of the few, as well as in vasculature ofovarian, endometrial and pancreatic tumors.

The invention will be further illustrated in the Examples that followsand which are not intended to limit the invention in any way.

Example Introduction

Compounds of the invention were made and tested as described below. Thefollowing disclosed embodiments are merely representative of theinvention which may be embodied in various forms. Thus, specificstructural, functional, and procedural details disclosed in thefollowing examples are not to be interpreted as limiting.

Materials and Methods Radiolabeling and QC

Labeling with ¹¹¹In

Indium (In-111) chloride in 50 mM HCl was purchased from MallinckrodtMedical B.V., Petten, The Netherlands, at an activity concentration of10-20 mCi/mL. In general, DOTA-peptide conjugates of the presentinvention were radiolabeled with Indium-111 at specific activities of0.1-0.2 mCi In-111/nmol DOTA-peptide conjugate. Briefly, 3-15 nmol ofDOTA-peptide conjugate dissolved in water was mixed with 2.5-12.5 μL of1.0 M pH 4.6 sodium acetate buffer, 1-5 μL of 0.1 M sodium ascorbate inwater and 30-150 μL of ¹¹¹InCl₃ (0.3-3.0 mCi). The radiolabelingreaction mixture was incubated in a boiling water bath for 20 to 30 min.For quality control a 2 μL aliquot of the radiolabeling solution wasquenched with 28 μL of an acetate buffered solution of Na₂-EDTA (5 mM,pH 4.6). After a successful radiolabeling (more than 95% peptide-boundradioactivity) Na₂-EDTA (0.1 M, pH 4.6) was added to the radiolabelingsolution to a final concentration of 1 mM.

Labeling with ⁶⁷Ga

Gallium (Ga-67) chloride was obtained either in dilute HCl at anactivity concentration of 498-743 mCi/mL from Nordion, Wesbrook Mall,Vancouver, Canada or at an activity concentration of 80 mCi/mL fromMallinckrodt Medical B.V., Petten, The Netherlands.

In general, DOTA-peptide conjugates of the present invention wereradiolabeled with Gallium-67 at specific activities of 0.1-0.2 mCiGa-67/nmol DOTA-peptide conjugate. Briefly, 3-15 nmol of DOTA-peptideconjugate dissolved in water was mixed with 50-125 μL of 1.0 M pH 4.0sodium acetate buffer and 5-15 μL of ⁶⁷GaCl₃ (0.5-3.0 mCi. Theradiolabeling reaction mixture was incubated in a boiling water bath for30 min. For HPLC quality control a 2 μL aliquot of the radiolabelingsolution was quenched with 28 μL of an acetate buffered solution ofNa₂-EDTA (5 mM, pH 4.0). After a successful labeling (more than 95%peptide-bound radioactivity) Na₂-EDTA (0.1 M, pH 4.0) was added to theradiolabeling solution to a final concentration of 1 mM.

Labeling with ¹⁷⁷Lu

Lutetium (Lu-177) chloride in 50 mM HCl was purchased from IDBRadiopharmacy, The Netherlands, at an activity concentration of 100mCi/mL.

In general, DOTA-peptide conjugates of the present invention wereradiolabeled with Lutetium-177 to a specific activity of up to 0.5 mCiLu-177/nmol DOTA-peptide conjugate. Briefly, 3-15 nmol of DOTA-peptideconjugate dissolved in water was mixed with 4-16 μL of 1.0 M pH 4.6sodium acetate buffer and 15-75 μL of ⁶⁷GaCl₃ (1.5-7.5 mCi). Radiolysiswas minimized by the addition of 5 μl of gentisic acid (80 mM) dissolvedin 0.2 M sodium ascorbate. The reaction mixture was incubated in aboiling water bath for 30 min. For HPLC quality control a 2 μL aliquotof the radiolabeling solution was quenched with 28 μL of an acetatebuffered solution of Na₂-EDTA (5 mM, pH 4.6). After a successfulradiolabeling (more than 95% peptide-bound radioactivity) Na₂-EDTA (0.1M, pH 4.6) was added to the radiolabeling solution to a finalconcentration of 1 mM.

Labeling with ^(99m)Tc

Tetraamine-coupled peptides were dissolved in 50 mM acetic acid/EtOH 8/2v/v to a final 1 mM peptide concentration. Each bulk solution wasdistributed in 50 μL aliquots in Eppendorf tubes and stored at −20° C.Labeling was conducted in an Eppendorf vial, wherein the followingsolutions were consecutively added: i) 0.5 M phosphate buffer pH 11.5(50 μL), ii) 0.1 M sodium citrate (5 μL, iii) [^(99m)Tc]NaTcO₄ generatoreluate (415 mL, 10-20 mCi), iv) peptide conjugate stock solution (15 μL,15 nmol) and v) freshly made SnCl₂ solution in EtOH (30 μg, 15 μL).After reaction for 30 min at ambient temperature, the pH was brought to7 by adding 1 M HCl (10 μL).

Quality Control

HPLC analyses were conducted on a Waters Chromatograph (Waters, Vienna,Austria) efficient with a 600 solvent delivery system; the chromatographwas coupled to twin detection instrumentation, comprising a photodiodearray UV detector (either Waters model 996 or model 2998) and a Gabigamma detector from Raytest (RSM Analytische Instrumente GmbH, Germany).Data processing and chromatography were controlled via the Millennium orEmpower 2 Software (Waters, USA). A XBridge Shield RP18 column (5 μm,4.6×150 mm, Waters, Ireland) coupled to the respective 2-cm guard columnwas eluted at 1 ml/min flow rate with a linear gradient system startingfrom 10% B and advancing to 70% B within 60 min, with solvent A=0.1%aqueous trifluoroacetic acid and solvent B=acetonitrile.

Metabolic Study in Mice Radioligand Injection and Blood Collection

A bolus containing the radioligand in normal saline (100-150 μL, =3nmol, 200-500 μCi) was injected in the tail vein of Swiss albino mice.Animals were kept for 5 min in a cage with access to water and were theneuthanized promptly by cardiac puncture while under a mild etheranesthesia. Blood (500-900 μL) was collected from the heart with asyringe and transferred in a pre-chilled Eppendorf tube on ice.

Plasma Separation and Sample Preparation

Blood was centrifuged to remove blood cells (10 min, 2000 g/4° C.). Theplasma was collected, mixed with acetonitrile (MeCN) in a 1/1 v/v ratioand centrifuged again (10 min, 15000 g/4° C.). Supernatants wereconcentrated to a small volume (gentle N₂-flux at 40° C.), diluted withsaline (z 400 μL) and filtered through a Millex GV filter (0.22 μm).

HPLC Analysis for Radiometabolite Detection

Aliquots of plasma samples (prepared as described above) were loaded ona Symmetry Shield RPM column which was eluted at a flow rate of 1.0mL/min with the following gradient: 100% A to 90% A in 10 min and from90% A to 60% for the next 60 min (A=0.1% aqueous TFA (v/v) and B=MeCN).Elution of radiocomponents was monitored by a gamma detector. For^(99m)Tc-radiopeptides, ITLC-SG analysis was performed in parallel usingacetone as the eluent to detect traces of TcO₄ ⁻ release (TcO₄ ⁻Rf=1.0).

Studies in GRPR⁺-Tumor Bearing Mice Tumor Induction

A 150 μL bolus containing a suspension of 1.5×10⁷ freshly harvestedhuman PC-3 cells in normal saline was subcutaneously injected in theflanks of female SCID mice. The animals were kept under asepticconditions and 2-3 weeks later developed well-palpable tumors at theinoculation site (80-150 mg).

Biodistribution and Calculation of Results

On the day of the experiment, the selected radiopeptide was injected inthe tail vein of tumor-bearing mice as a 100 μL bolus (1-2 μCi, 10 pmoltotal peptide; in saline/EtOH 9/1 v/v). Animals were sacrificed ingroups of four under a mild ether anesthesia by cardiac puncture atpredetermined time points pi (postinjection). Additional sets of threeto four animals were co-injected with excess [Tyr⁴]BBN(=40 nmol) alongwith test radiopeptide and were sacrificed at 4 h pi (blocked animals).Samples of blood and tissues of interest were immediately collected,weighed and measured for radioactivity in a γ-counter. Stomach andintestines were not emptied of their contents, but measured ascollected. Biodistribution data were calculated as percent injected doseper gram tissue (% ID/g) using the Microsoft Excel program with the aidof suitable standards of the injected dose.

Results

The results of the various illustrative tests are described herebelow byreferring to the corresponding figure. Specific structural, functional,and procedural details disclosed in the following results are not to beinterpreted as limiting.

FIG. 1A: Biodistribution of [¹¹¹In]NeoBOMB-1(¹¹¹In-DOTA-(p-aminobenzylamine-diglycolicacid)-[DPhe⁶,His¹²-CO—NH—CH[(CH₂CH(CH₃)₂)₂,des-Leu¹³,des-Met¹⁴]BBN(6-14))in female SCID mice bearing PC-3 tumors (hGRPR⁺) at 4 h and 24 h pi.Bars represent average uptake as % injected does per gram (% ID/g) of atleast 4 animals with standard deviation; an additional group of animalsreceived excess [Tyr⁴]BBN (100 μg) for in vivo receptor blockade at 4 hpi. Bl=blood, Li=liver, He=heart, Ki=kidneys, St=stomach, In=intestines,Sp=spleen, Mu=muscle, Lu=lungs, Pa=pancrease, Fe=femur and Tu=PC-3tumor. High uptake and retention is observed in the experimental tumorwith 28.6±6.0% ID/g at 4 h and 25.9±6.6% ID/g at 24 h. A high percentageof this uptake could be significantly reduced by co-injection of excessof a native bombesin analog.

FIG. 1B: Radiochromatogram of ex-vivo mouse blood 5 min after injectionof [¹¹¹In]NeoBOMB-1. The percentage of parent peptide remaining intactis >91%.

FIG. 1C: Biodistribution of [¹⁷⁷Lu]NeoBOMB-1(¹⁷⁷Lu-DOTA-(p-aminobenzylamine-diglycolicacid)-[DPhe⁶,His¹²-CO—NH—CH[(CH₂CH(CH₃)₂]₂,des-Leu¹³,des-Met¹⁴]BBN(6-14))in female SCID mice bearing PC-3 tumors (hGRPR⁺) at 4, 24 and 72 h pi.Bars represent average uptake as % injected dose per gram (% ID/g) of atleast 4 animals with standard deviation; an additional group of animalsreceived excess [Tyr⁴]BBN (100 μg) for in vivo receptor blockade at 4 hpi. Bl=blood, Li=liver, He=heart, Ki=kidneys, St=stomach, In=intestines,Sp=spleen, Mu=muscle, Lu=lungs, Pa=pancrease, Fe=femur and Tu=PC-3tumor. Pancreatic uptake declines more rapidly with time than tumoruptake resulting in increasingly higher tumor-to-pancreas ratios,especially at 72 h pi.

FIG. 1D: Radiochromatogram of ex-vivo mouse blood 5 min after injectionof [¹⁷⁷Lu]NeoBOMB-1, shows that >92% parent peptide remains intact.

FIG. 1E: Biodistribution of [⁶⁷Ga]NeoBOMB-1(⁶⁷Ga-DOTA-(p-aminobenzylamine-diglycolicacid)-[DPhe⁶,His¹²-CO—NH—CH[(CH₂CH(CH₃)₂]₂,des-Leu³, des-Met⁴]BBN(6-14))in female SCID mice bearing PC-3 tumors (hGRPR⁺) at 1 h and 4 h pi. Barsrepresent average uptake as % injected does per gram (% ID/g) of atleast 4 animals with standard deviation; an additional group of animalsreceived excess [Tyr⁴]BBN (100 μg) for in vivo receptor blockage at 4 hpi. Bl=blood, Li=liver, He=heart, Ki=kidneys, St=stomach, In=intestines,Sp=spleen, Mu=muscle, Lu=lungs, Pa=pancreas, Fe=femur and Tu=PC-3 tumor.High tumor values (>30% ID/g) are achieved by the radiotracer at 1 and 4h pi.

FIG. 1F: Radiochromatogram of ex-vivo mouse blood 5 min after injectionof [⁶⁷Ga]NeoBOMB-1, shows that >97% parent peptide remains intact.

FIG. 2A: Biodistribution of [^(99m)Tc]NeoBOMB-2(^(99m)Tc—N₄-(p-aminobenzylamine-diglycolicacid)-[DPhe⁶,His¹²-CO—NH—CH[(CH₂—CH(CH₃)₂]₂,des-Leu³,des-Met¹⁴]BBN(6-14))in female SCID mice bearing PC-3 tumors (hGRPR⁺) at 1 h, 4 h and 24 hpi. Bars represent average uptake as % injected dose per gram (% ID/g)of at least 4 animals with standard deviation; an additional group ofanimals received excess [Tyr⁴]BBN (100 μg) for in vivo receptor blockadeat 4 h pi. Bl=blood, Li=liver, He=heart, Ki=kidneys, St=stomach,In=intestines, Sp=spleen, Mu=muscle, Lu=lungs, Pa=pancreas and Tu=PC-3tumor. High tumor values (˜30% ID/g) are achieved by the ratiotracer at1 and 4 h pi, which remain exceptionally high (>25% ID/g) at 24 h pi.

FIG. 2B: Radiochromatogram of ex-vivo mouse blood 5 min after injectionof [^(99m)Tc]NeoBOMB-2 shows that >88% parent peptide remains intact.

1-19. (canceled)
 20. A method of diagnosing and/or treating primaryand/or metastatic GRPR-positive cancer, the method comprisingadministering an effective amount of a radiolabeled GRPR-antagonist to asubject, wherein said radiolabeled GRPR-antagonist is of the generalformula MC—S—P wherein: M is a radiometal and C is a metal chelator thatstably binds M, or MC is a Tyr- or prosthetic group bound to aradiohalogen, S is a spacer covalently linked between C and P, wherein Sis covalently attached to the N-terminus of P; and P isDPhe-Gln-Trp-Ala-Val-Gly-His-NH—CH[CH₂—CH(CH₃)₂]₂ (SEQ ID NO: 1). 21.The method as claimed in claim 20, wherein S is selected from the groupconsisting of compounds of the formulae:


22. The method as claimed in claim 20, wherein S is:


23. The method as claimed in claim 20, wherein M is selected from thegroup consisting of ¹¹¹In, ^(99m)Tc, ^(94m)Tc, ⁶⁷Ga, ⁶⁶Ga, ⁶⁸Ga, ⁵²Fe,¹⁶⁹Er, ⁷²As, ⁹⁷Ru, ²⁰³Pb, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ¹⁸⁶Re, ¹⁸⁸Re, ⁸⁶Y, ⁹⁰Y,⁵¹Cr, ^(52m)Mn, ¹⁵⁷Gd, ¹⁷⁷Lu, ¹⁶¹Tb, ¹⁶⁹Yb, ¹⁷⁵Yb, l⁰⁵Rh, ¹⁶⁶Dy, ¹⁶⁶Ho,¹⁵³Sm, ¹⁴⁹Pm, ¹⁵¹Pm, ¹⁷²Tm, ¹²¹Sn, ^(117m)Sn, ²¹³Bi, ¹⁴²Pr, ¹⁴³Pr,¹⁹⁸Au, ¹⁹⁹Au, ¹²³I, ¹²⁴I, ¹²⁵I, and ¹⁸F.
 24. The method as claimed inclaim 20, wherein the metal chelator C is a metal chelator for di- andtrivalent metals.
 25. The method as claimed in claim 23, wherein themetal chelator for di- and trivalent metals is a DTPA-, NOTA-, DOTA-, orTETA chelator or a derivative thereof, including bifunctional derivativethereof.
 26. The method as claimed in claim 20, wherein the metalchelator C is obtained by grafting to the spacer S, a metal chelatorselected from the group consisting of:


27. The method as claimed in claim 20, wherein the metal chelator C is ametal chelator for radionuclides of technetium or rhenium.
 28. Themethod of claim 20, wherein C is a metal chelator for radionuclides oftechnetium or rhenium selected from the group consisting of acyclictetraamine; cyclam; PnAO; tetradentate chelators containing donor atomsets selected from the group consisting of P₂S₂—, N₂S₂— and N₃S—;derivatives of said tetradentate chelators; bifunctional derivatives ofsaid tetradentate chelators; HYNIC/co-ligand-based chelators; and bi-and tridentate chelators that form organometallic complexes viatricarbonyl technology.
 29. The method as claimed in claim 20, whereinthe metal chelator is obtained by grafting to the spacer S a chelatorfor radionuclides of technetium or rhenium selected from the groupconsisting of:

and bifunctional derivatives thereof.
 30. The method of claim 20,wherein the cancer is selected from prostate cancer, breast cancer,small cell lung cancer, colon carcinoma, gastrointestinal stromaltumors, gastrinoma, renal cell carcinomas, gastroenteropancreaticneuroendocrine tumors, oesophageal squamous cell tumors, neuroblastomas,head and neck squamous cell carcinomas, as well as in ovarian,endometrial and pancreatic tumors displaying neoplasia-relatedvasculature that is GRPR-positive.
 31. The method of claim 20, whereinthe subject is human.
 32. The method of claim 20, wherein the M is¹¹¹In, ¹⁷⁷Lu, ⁶⁷Ga, ⁶⁸Ga, ^(99m)Tc, ¹⁸⁶Re, or ¹⁸⁸Re.
 33. The method ofclaim 20, wherein M is ¹¹¹In, ¹⁷⁷Lu, ⁶⁷Ga, or ⁶⁸Ga.
 34. The method ofclaim 20, wherein M is ^(99m)Tc, ¹⁸⁶Re, or ¹⁸⁸Re.
 35. The method ofclaim 20, wherein M is a radiohalogen ¹⁸F.